We have taken a novel approach to antiviral drug discovery, using cell-free protein synthesis to recreate the host-catalyzed capsid assembly pathways for 20 of the 23 families of viruses causing human disease. Ten of these have been adapted to moderate throughput whole pathway screens by which small molecules that interfere with protein-protein interactions in this pathway can be identified. Using this approach a plethora of distinct pharmacophores with potent activity against infectious virus in cell culture has been identified, for every viral family studied. Advanced analogs reveal substantial improvement in potency with concomitant diminution in toxicity, suggesting distinct structure-activity relationships (SAR) for efficacy and toxicity. The advanced analogs have also been used as ligands for affinity chromatography to identify the host targets. These host targets have been shown to comprise assembly machines that show a remarkable degree of viral specificity: they appear to be modified by the nature of the substrate being assembled, hence accounting for our ability to target their use by the virus, without significant toxicity to the host. This has resultd in identification of numerous lead series' with selectivity indices (CC50/EC50, SI) > 100. The advanced compounds display some unusual properties consistent with their activity against host targets: in general, they are effective against all members of a viral family, with no evidence of viral resistance development. Here we propose to advance a small molecule lead series identified in a cell-free whole pathway screen for rabies virus (RABV) capsid assembly that has potent activity against infectious RABV in cell culture. SAR will be used in the R21 phase of the project to achieve modest advances in potency and diminution of toxicity necessary to improve the SI from 50-100 to >100. We will also optimize pharmacokinetic and pharmacodynamic (PK/PD) properties sufficient to justify advancement under the R33 component to proof of concept animal efficacy testing in a pre-exposure model using mice. Upon success, further R33 studies will advance the most efficacious compounds to post-exposure prophylaxis in the hamster model of RABV infection, with additional lead optimization, as needed. The proposed work will advance RABV small molecule therapeutics by achieving animal efficacy in two vertebrate species, with adequate exposure to support once or twice daily dosing, thereby setting the stage for future IND-enabling studies.